Helicases are mechanochemical enzymes that couple the energy of nucleoside triphosphate hydrolysis to the dehybridization or unwinding of duplex nucleic acid molecules (1). Nucleic acid unwinding is of central importance in a variety of nucleic acid transactions that include the transcription, translation, recombination, and replication of genetic material. The importance of helicases is further underscored by the large number of DNA or RNA helicases identified in prokaryotic and eukaryotic organisms.
Assays that are both rapid and quantitative for the routine measurement of helicase activity have not been available. There have been essentially two types of helicase assays developed to date. One type of assay has relied on the generation of single-stranded, nuclease sensitive molecules from nuclease insensitive, uniformly radiolabeled double-stranded substrates, followed by the quantitation of acid soluble nucleotides (2). Alternatively, helicase activity has been determined by the unwinding of model duplex substrates comprised of a labeled oligonucleotide hybridized to a larger unlabeled single-stranded molecule followed by size separation (3, 4). Because of the difficulty in obtaining assay substrates suitable for performing the first type of assay, the latter assay has been more commonly utilized. The use of model duplex substrates has also generated the greater variety of nucleic acid structures necessary for the detection of activity associated with different classes of helicase enzymes.
Although the construction of model helicase substrates is useful for detecting enzyme-associated helicase activity, the measurement of that activity is unduly tedious when large numbers of assays need to be performed. This is due to the methods commonly employed in the quantitation of helicase reaction products. Helicase reaction products have primarily been separated from substrates by non-denaturing PAGE (4). Alternative methods have included the hybridization of single-stranded helicase-generated nucleic acid molecules to scintillant-containing beads or fluorescence methods based on resonance energy transfer properties of labeled nucleic acid molecules or specific double-stranded DNA dyes (5-8). In one recently published solution phase helicase assay, DNA intercalators were used as an indicator fluorescent dye for double-stranded DNA molecules (8). In that assay, helicase activity was measured by a decrease in fluorescence.
Each of the above-described quantitative methods suffers from individual drawbacks in terms of accuracy, reproducibility, use of special or sophisticated equipment, time required, expense and capacity to measure routinely large numbers of samples for helicase activity.